Regeneration of Spinal Discs

ABSTRACT

Compositions and processes for the effective and efficient regeneration of spinal discs are provided. These compositions contain stem cells, donor cells, and platelet plasma compositions. By using these compositions, there is an increased likelihood of acceptance and proper cell differentiation.

FIELD OF THE INVENTION

The present invention relates to the field of regeneration of spinaldiscs.

BACKGROUND OF THE INVENTION

One of the major causes of back pain and disability is degeneration oflumbar intervertebral discs. This source of pain may affect up to 80% ofAmericans over the course of their lifetimes. Furthermore, the economicimpact of treating persons who suffer from disc degeneration is over $20billion per year. Thus, disc degeneration has a significant effect on asizeable portion of the population.

Disc degeneration may begin in childhood as cells within anintervertebral disc that produce the substrates that maintain dischydration undergo normal senescence. At approximately the time that aperson is four years old, his or her notochord cells are lostpermanently, and their function is replaced by cells calledchondrocytes. Both chondrocytes and notochord cells produceproteoglycans; however, chondrocytes also produce significant amounts ofcollagen, thereby causing discs to become hydrated and firmer. Thisphysiologic event marks the onset of disc degeneration.

Disc degeneration may be accelerated by several factors, including butnot limited to injury and aging. Ideally, discs would regenerate ontheir own. Unfortunately, the intervertebral discs of the spine oftenfail to heal from injury and aging because of a paucity of bloodvessels. The lack of a robust blood supply causes the intervertebraldiscs to obtain nutrition and to eliminate waste through the process ofimbibition, which is the displacement of one fluid by another fluid thatis immiscible. Over time, this process causes a lower pH to develop inthe intervertebral discs and homeostasis via imbibition cannot besustained.

When there is disc degeneration, patients can experience significantpain, and researchers and clinicians have long sought methodologies fortreating this pain. One of the common historical approaches has beenspinal fusion, which is the joining of two or more vertebrae. Spinalfusion addresses the degenerating disc by excision of the disc andsubsequently, either fusing the spine with bone products or replacingthe excised disc with a mechanical device.

Unfortunately, spinal fusions can cost between $60,000 and $100,000 perpatient and can be associated with complications at twice the rate ofcomplications that accompany nerve decompression surgery, which isanother method for treating pain. Additionally, the result of spinalfusion surgery is a shift of mechanical stress to the intervertebraldisc level above and/or below the fused or replaced disc. This commonphenomenon is known as adjacent level disease/degeneration, and it toocan contribute to discomfort in a patient. A further challenge fortreating compromised discs by the common methods used by practitionersis that these methods are primarily non-biologic in nature, and thusface challenges in being accepted by the recipient.

Because of the high costs that are associated with lumbar fusions alongwith inconsistent outcomes and higher complication rates, researchersand clinicians have developed biologic and other non-fusion approachesfor treating disc degeneration. Current non-fusion approaches fortreating a degenerating disc include stabilizing the disc, rehydratingthe disc, rebalancing the pH in the disc, intradiscal injections,provision of extracellular matrix proteins, intradiscal pressurereduction, disc denervation, disc nucleus replacement, stimulation ofdisc chondrocyte proliferation, non-ablation laser treatment and stemcell therapy. However, none of these strategies as currently employedprovide optimally effective and efficient means for treating degenerateddiscs. Thus, there is a need to develop new means by which to addressdisc degeneration.

SUMMARY OF THE INVENTION

The present invention provides compositions for treating degeneratedspinal discs, as well as processes for making and using thesecompositions. Through the use of various embodiments of the presentinvention, one can increase the ability of discs to be regenerated andthereby reduce pain in patients who suffer from disc degeneration.

According to a first embodiment, the present invention is directed to acomposition for regenerating a disc. The composition comprises: (a) stemcells from a subject; (b) donor disc cells, wherein the donor disc cellsare derived from a non-degenerative level of a spine of the subject; and(c) a platelet plasma composition. In some embodiments, the donor disccells are chondrocytes. Additionally, in some embodiments, thecomposition further comprises one or more of an extra-cellular matrixthat, for example, may be derived from the non-degenerative level of thespine, growth factors such as platelet-derived growth factors, calciumchloride or a combination of calcium chloride and thrombin.

Optionally, prior to forming the composition for disc regeneration, onemay morselize and/or extrude the chondrocytes. Further, prior to formingthe composition for disc regeneration, one may concentrate orhemoconcentrate bone marrow aspirate to form a solution containing thestem cells and/or concentrate or hemoconcentrate the entire composition.The term “composition” means any combination of two or more substances,including, but not limited to, a mixture, a suspension, or a solution.

Furthermore, in some embodiments, the composition for disc regenerationis photoactivated. For example, it may be incubated under visible lightwavelengths in order to increase the efficiency of the differentiationof the stem cells.

According to a second embodiment, the present invention is directed to aprocess for creating a composition for disc regeneration. The processcomprises: (a) morselizing donor disc material, wherein the donor discmaterial comprises healthy cells obtained from a non-degenerative levelof a spine; (b) extruding the donor disc material to form an extrudedmaterial that comprises chondrocytes and an extracellular matrix; and(c) mixing the chondrocytes, the extra cellular matrix, a bone marrowaspirate and a platelet plasma composition, wherein the platelet plasmacomposition comprises growth factors to form a composition for discregeneration.

According to a third embodiment, the present invention is directed to aprocess for regenerating a disc. The process comprises: (a) harvestingbone marrow from a person; (b) generating bone marrow aspirate and aplatelet plasma composition from the bone marrow, wherein the bonemarrow aspirate comprises mesenchymal stem cells, plasma, platelets andred blood cells; (c) harvesting disc chondrocytes and an extra cellularmatrix from non-degenerative disc tissue of the person; and (d)combining the bone marrow aspirate and the disc chondrocytes to form acomposition under conditions that permit the mesenchymal stem cells tobegin differentiation.

According to a fourth embodiment, the present invention provides amethod for treating disc degeneration comprising administering acomposition of the present invention to a person in need thereof.Preferably, a person of ordinary skill in the art will administer atherapeutically effective amount of the composition.

According to a fifth embodiment, the present invention provides a methodtreating disc degeneration comprising administering a composition madeby a process of one or more of the embodiments of the present inventionand further comprising: (a) mechanically decompressing the recipientdisc; and (b) injecting the mixture into the recipient disc. Theinjection may be into the disc nucleus pulposus and/or into theposterior/posterolateral annulus fibrosus. Optionally, prior toinjecting the mixture one may apply a low level laser treatment to therecipient disc.

Additionally, in various embodiments, a material for forming a scaffoldmay be used. This material may for example comprise bone marrowaspirate, a platelet plasma composition from the bone marrow and acoagulant that is made from CaCl₂ and thrombin. One may combine thescaffolding material with the composition prior to administration, orone may apply it to the site of interest after the composition isapplied, or one may apply it to the site of interest prior toapplication of the composition.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to various embodiments of thepresent invention. In the following detailed description, numerousspecific details are set forth in order to provide a thoroughunderstanding of the present invention. However, unless otherwiseindicated or implicit from context, the details are intended to beexamples and should not be deemed to limit the scope of the invention inany way.

According to one embodiment, the present invention is directed to acomposition for regenerating a disc in need thereof. The disc that needsregeneration may be referred to as a “target disc.” The target disc maybe in need of regeneration because of degeneration due to aging ortrauma or a combination thereof and may or may not be a source of painfor a patient. Alternatively or additionally, it may be in need ofregeneration because of herniation. As persons of ordinary skill in theart will recognize, a target disc may be partially or completelydegenerated.

The composition for regeneration may, for example, comprise, consistessentially of or consist of stem cells, donor disc cells, and aplatelet plasma composition. The components may be combined prior toadministration to a site of interest (i.e., the target disc) or they maybe combined at the site of interest. When the composition is formedprior to introduction into a patient, it may be in the form of amedicament for use in treating disc degeneration. In some embodiments,all of the stem cells, the donor disc cells and the platelet plasmacomposition are derived from the same subject. In other embodiments,only the stem cells and the donor disc cells are derived from the samesubject. The subject may, for example, be a human or other mammal, andpreferably is the patient in need of disc regeneration.

Stem Cells

Stem cells are cells that have the capability to differentiate into aplurality of different types of cells. Thus, they can be replacementcells or a source of replacement cells, and they are capable ofreplacing cells that die due to injury, illness or disease. Cell deathis referred to as senescence.

The path of differentiation that stem cells take is strongly influencedby cell-to-cell signaling. If stem cells are placed in a muscle, theycan differentiate into muscle cells. If stem cells are placed in atendon, they can differentiate into tendon cells. If one engraftsmesenchymal stem cells into a disc, the mesenchymal stem cells willdifferentiate into chondrocytes. However, if one were to place stemcells directly into an environment in which there is alreadydegeneration, there is a significant likelihood that they would developinto degenerative type cells. Accordingly, and as discussed in moredetail below, in various embodiments of the present invention oneexposes stem cells to donor disc cells that are from a non-degenerativelayer of the spine prior to injecting the stem cells into a target site.Preferably, both the stem cells and the donor disc cells are from thesame subject.

Various embodiments of the present invention use stem cells for one ormore the following purposes: (1) to stimulate natural disc regeneration;(2) to reduce inflammation within an intervertebral disc, epidural spaceand/or facet joint; (3) to provide replacement cells for senescentintervertebral disc cells; (4) to replenish an extracellular matrix; (5)to reduce pain; (6) to restore/maintain intervertebral disc height; (7)to stabilize intervertebral discs; and (8) to attract additional stemcells through chemotaxis.

Examples of stem cells that may be used in connection with the presentinvention are those that are derived from bone-marrow as well as thosethat are derived from adipose tissue, (e.g., subcutaneous fat), synovialfluid, peripheral blood, and amniotic fluid or amniotic membrane. Thecomposition for regeneration may contain stem cells from any of theseindividual sources or from any plurality of these sources. Preferablythe mesenchymal stem cells that are used are autologous. The term“autologous” means being derived from the same individual's body intowhich they will be transferred. By being autologous, there is asignificantly reduced risk of rejection.

As persons or ordinary skill in the art know, within human bone marrow,there are a number of different types of blood cells, includingmesenchymal stem cells. These cells are multipotent and candifferentiate into a variety of cell types, including osteoblasts,chondrocytes and adipocytes. When obtaining mesenchymal stem cells fromthe bone marrow, one may directly remove a quantity of bone marrow intovarious size syringes that contain a pre-measured amount ofanticoagulant. In some embodiments, 20-2000 cc or 50-1000 cc or 60-500cc of bone marrow per ilium is extracted from the pelvis or 20-500 cc or30-250 cc or 50-100 cc of bone marrow is retrieved per side ofvertebrae. Examples of anticoagulants include but are not limited toEDTA, citrate, oxalate, ACD (acid citrate dextrose, which also may bereferred to as anticoagulant citrate dextrose) in the form of ACD-A orACD-B and heparin, which may be used in pre-coated syringes. By way of anon-limiting example, the volume of bone marrow may be between eight andten times the volume of the anticoagulant, e.g., 54 cc of bone marrowand 6 cc of ACD-A.

In some embodiments, ultimately between 2 cc and 6 cc or between 3 ccand 5 cc or between 3.5 cc and 4.5 cc of BMAC is inserted to regeneratea disc. As persons of ordinary skill in the art will recognize the exactvolume will depend on the size of the disc in need of regeneration orhealing, which in part depends on the size of the patient.

After removal of the bone marrow, one may subject the contents of thesyringe to centrifugation, thereby stratifying the bone marrow intospecific blood tissue layers, e.g., bone marrow aspirate concentrate(BMAC) and a platelet plasma composition. The mesenchymal stem cells,which are part of the BMAC may then be removed for use in variousembodiments of the present invention. Processes for removing products bycentrifugation are well known to persons of ordinary skill in the art.

As noted above, stem cells may also be derived from adipose tissue.Human adipose tissue is a well-vascularized tissue, and the network ofblood vessels that are integrated throughout subcutaneous fat issurrounded by numerous types of regenerative cells. These regenerativecells include mesenchymal stem cells as well as other cells that areimportant for tissue healing and regeneration such as monocytes andfibroblasts.

Cells that are positioned around the blood vessels in adipose tissue arecommonly referred to as pericytes. The fraction of regenerativepericytes in adipose tissue is known as the stromal vascular fraction(SVF).

In order to obtain adipose tissue, one may harvest fat from the abdomenor the flank. After harvesting, one may separate SVF from the cells. Byway of non-limiting examples, one may isolate SVF from the fat cellsutilizing ex vivo ultrasonic cavitation, in vivo mechanical separation,collagenase digestion or lecithin emulsification. When isolating theSVF, one may spin down the material in order to obtain a pellet.Preferably, the pellet is substantially free of collagen.

By looking to adipose tissue for mesenchymal stem cells, one is able toyield a greater amount of mesenchymal stem cells than one is able toobtain from bone marrow. This benefit of using adipose tissue may berealized regardless of the age of the person from whom the tissue isobtained. Thus, in some embodiments, one may use stem cells exclusivelyfrom bone marrow, exclusively from fat tissue or stem cells from acombination thereof. When stem cells from a plurality of sources areused in the same composition, one may use them in approximately equalvolume or in substantially unequal volumes, for example, 40-60% of eachsource or in substantially unequal volumes of 20-40% of adipose derivedstem cells and 60-80% of bone marrow derived stem cells or 60-80%adipose derived stem cells and 20-40% of bone marrow derived stem cells.In one non-limiting example, a person of ordinary skill in the art maycombine a pellet or ½ of a pellet of stem cells from adipose tissue witha volume of 1 cc-5 cc or 3 cc-5 cc or 1.5 cc-3 cc of BMAC.

Donor Disc Cells: Disc Chondrocytes

Donor disc cells are cells that are derived from a disc that isnon-degenerative. The term “non-degenerative” means that it is not asite that is to be regenerated. Preferably, there is no degeneration orno degeneration beyond the minimal amount due to normal aging. Anon-degenerative site may also be defined as a site with lessdegeneration than a site to which stem cells will be introduced.

In various embodiments of the present invention, the donor disc cellsserve one or more, if not all, of the following purposes: (1) to providenon-degenerative intradiscal cells for autologous mesenchymal stem cellsfor socialization/differentiation; (2) to reduce inflammation within theintervertebral disc; (3) to provide replacement cells for senescentintervertebral disc cells; (4) to replenish extracellular matrix; (5) toreduce pain; (6) to restore/maintain intervertebral disc height; (7) tostabilize intervertebral discs; (8) to attract additional stem cellsthrough chemotaxis; and (9) to add a biologic scaffold.

In order to obtain donor disc cells, one may introduce a guide wire intoa site at which to harvest using fluoroscopic x-rays for guidance. Onemay then make an incision and use a retractor or cannula. The guide wireis removed, and one may then harvest donor disc material that comprisesthe donor disc cells. Examples of tools that may be used to harvestthese materials include but are not limited to, an endopituitary tool, awater jet, a suction punch or other mechanical means. One may harvestthe donor disc material from the nucleus pulposus and/or annulusfibrosis.

Examples of sites from which to obtain donor disc material include butare not limited to the sacral level (S1/2), the thoracic level, thelumbar level, the stress shielded normal variant disc and anon-degenerative part of the treatment level. By way of a non-limitingexample, in order to regenerate a disc, one may extract approximately1-100 or 2-50 or 2-20 or 5-10 pieces of donor disc material that eachare between 0.5-10 mm³ or 0.5-2 mm³ (e.g., approximately 1 mm³) in size,and morselize them down to smaller sized pieces to put into discs. Thesmaller pieces may comprise, consist essentially of or consist of singlecells or small clumps of cells, protein strands and connective tissue.

In some embodiments, the donor disc material is processed prior tomixing with the stem cells. For example, one may morselize the donordisc material in order to increase the surface area for contacts betweenstem cells and normal discs. Morselization will also expand the volumeof normal extracellular matrix, which is part of the retrieved donordisc material. Methods for morselizing may, for example, employ ascalpel, a water jet and a morselizer.

Following or instead of morselization, the donor disc material may besubjected to extrusion. Extrusion frees chondrocytes from theirsurrounding matrix (e.g., an extracellular matrix, which is alsoreferred to as ECM, and contains the interstitial matrix and thebasement membranes). As with morselization, extrusion increases the cellsurface in order to optimize contact between stem cells and normal disccells.

In one non-limiting example, the extrusion process is accomplishedthrough the use of two syringes and a connector. The connector has asmaller bore than the syringes. When the donor disc material is sentback and forth between the syringes, turbulence is created. Theturbulence forces cells away from the other components of the tissue andcan, in some embodiments, break cells apart.

Platelet Plasma Composition

The platelet plasma composition comprises, consists essentially of, orconsists of platelets and plasma and may be derived from bone marrow orperipheral blood. The present invention may use platelet plasmacompositions from either or both of these sources, and either plateletplasma composition may be used to regenerate either a nucleus or annulusin need thereof. Further, the platelet plasma composition may be usedwith or without concentrated bone marrow (BMAC). By way of example, wheninserted into the annulus, 0.05-2.0 cc of platelet plasma compositionmay be used, and when inserted into the nucleus, 0.05-3.0 cc of theplatelet plasma composition may be used.

Platelets are non-nucleated blood cells that as noted above are found inbone marrow and peripheral blood. They have several important functionssuch as controlling bleeding and tissue healing. As persons of ordinaryskill in the art are aware, the ability to promote tissue healing is dueto the many growth factors that they produce including platelet-derivedgrowth factor (PDGF), transforming growth factor beta (TGF-beta),fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1),connective tissue growth factor (CTGF) and vascular endothelial growthfactor (VEGF). Many of these platelet proteins and molecules arecytokines and are important for cell signaling and immunomodulation.

In various embodiments of the present invention, the platelet plasmacomposition may be obtained by sequestering platelets from whole bloodor bone marrow through centrifugation into three strata: (1) plateletrich plasma; (2) platelet poor plasma; and (3) fibrinogen. When usingplatelets from one of the strata, e.g., the platelet rich plasma (PRP)from blood, one may use the platelets whole or their contents may beextracted and concentrated into a platelet lysate through a cellmembrane lysis procedure using thrombin and/or calcium chloride. Whenchoosing whether to use the platelets whole or as a lysate, one mayconsider the rate at which one desires regeneration and/or tissuehealing (which may include the formation of scar tissue withoutregeneration or healing of a herniated or torn disc). In someembodiments the lysate will act more rapidly than the PRP (or plateletpoor plasma from bone marrow).

Notably, platelet poor plasma that is derived from bone marrow has agreater platelet concentration than platelet rich plasma from blood,also known as platelet poor/rich plasma, (“PP/RP” or “PPP”). PP/RP orPPP may be used to refer to platelet poor plasma derived from bonemarrow, and in some embodiments, preferably PP/RP is used or PRP is usedas part of the composition for disc regeneration. (By convention, theabbreviation PRP refers only to compositions derived from peripheralblood and PPP (or PP/RP) refers to compositions derived from bonemarrow.)

In various embodiments, the platelet plasma composition, which may ormay not be in the form of a lysate, may serve one or more of thefollowing functions: (1) to release/provide growth factors and cytokinesfor tissue regeneration; (2) to reduce inflammation; (3) toattract/mobilize cell signaling; (4) to initiate fibroblast repair ofdamaged annulus through fibroblast growth factors (FGF); (5) tostabilize disc annulus; (6) to repair annulus disc tears; (7) tostimulate revascularization to a disc; and (8) to stimulate stem cellactivation. Additionally, by combining platelet therapy with stem cells,there can be synergy with respect to reducing back pain.

In some embodiments in which the lysate is used, the cytokines areconcentrated in order to optimize their functional capacity.Concentration may be accomplished in two steps. First, blood may beobtained and concentrated to a volume that is 5-15% of what it wasbefore concentration. Devices that may be used include but are notlimited to a hemofilter or a hemoconcentrator. For example, 60 cc ofblood may be concentrated down to 6 cc. Next, the concentrated blood maybe filtered to remove water. This filtering step may reduce the volumefurther to 33%-67% (e.g., approximately 50%) of what it was prior tofiltration. Thus, by way of example for a concentration product of 6 cc,one may filter out water so that one obtains a product of approximately3 cc.

When the platelet rich plasma, platelet poor plasma and fibrinogen areobtained from blood, they may for example be obtained by drawing 20-500cc of peripheral blood, 40-250 cc of peripheral blood or 60-100 cc ofperipheral blood. The amount of blood that one should draw will dependon the number of discs that have degenerated and the size of the discs.As persons of ordinary skill in the art will appreciate, a typical dischas a volume of 2-5 cc or 3-4 cc.

By way of non-limiting example, for a given disc, one may use acomposition that comprises, consists essentially of or consists of ½-1cc of PPP, 3-5 cc of BMAC and 10 mm³ donor disc cells.

Composition for Regeneration

In some embodiments, prior to injecting the stem cells into thedegenerative disc, one mixes them with donor disc cells forapproximately 10-40 minutes or 20-40 minutes. In some embodiments, thestem cells are part of the BMAC at the time of combination.Additionally, preferably mixing is accompanied by warming

Next, one may mix the donor disc cells and ECM with BMAC (if not alreadypresent or optionally with more BMAC if already present) and theplatelet plasma composition, which preferably comprises growth factors.By mixing the donor disc cells with stem cells, one may initiate stemcell differentiation down a chondrocyte cell line through socialization,due to cell-to-cell signaling. The growth factors may, for example, bethose identified elsewhere in this disclosure or that are now known orthat come to be known to persons of ordinary skill in the art and thatwould be recognized as of use in connection with the present invention.Furthermore, the mixture may contain calcium chloride or both calciumchloride and thrombin to release growth factors. Mixing may beaccomplished through the use of mechanical devices that are designed topermit mixing of the aforementioned materials, and be done at roomtemperature or warmed to a temperature greater than room temperature,but below which undesirable denaturation of proteins would occur.Optionally, it may also be activated under light.

Photoactivation

In some embodiments, one photoactivates the concentrated BMAC and donorchondrocytes. Optionally, this mixture also contains PP/RP or PRP at thetime of photoactivation. Photoactivation may be carried out by amonochromatic light source, and photoactivation may take place prior toor after introducing the stem cells to the site of interest for a periodof 10 minutes to 2 hours or 15 minutes to 1 hour or 20 minutes to 40minutes.

In some embodiments, one carries out photoactivation under a wavelengthof 390 nm-900 nm or 390 nm-600 nm, or 600 nm-900 nm or 600 nm-750 nmPhotoactivation may reduce pain, promote healing and/or increase theability of stem cells to differentiate. In some embodiments, the mixtureis incubated after it contains all ingredients but prior toadministration. For example, one may use an Adi-Light or Adi-Light 2.Photoactivation increases the presence of the Interleukin-1 ReceptorAntagonist (IL-1RA), which decreases the pain and inflammationassociated with injections.

Target Site

The target site is the disc location at which there is degenerationand/or herniation. Optionally, prior to introduction of the stem cells,one decompresses the discs at the target site, which may also be knownas the recipient disc. This may, for example, be accomplished bymechanical means. In order to cause mechanical decompression, one may,for example, use an endopituitary tool. Decompression both producesrelief of pressure and creates space for the composition forregeneration and/or channels for rehydration.

Laser Therapy

In various embodiments of the present invention, one may also use lasertherapy. The laser may, for example, be used to treat the nucleuspulposus of a disc of interest, and the process of using the laser maybe referred to as photostimulation. When employing laser therapy onemay, for example, use a Holmium:YAG; Nd:YAG; and/or Erbium:YAG laserlight at non-ablation settings. By using the laser treatment atnon-ablation settings one can stimulate chondrocyte proliferation anddisc rehydration by a plurality of methods.

First, the non-ablation laser generator settings result in onlylow-level heat production and not thermal ablation of the disc. Thus,the low level heat production is able to stimulate chondrocyteproliferation without significant protein denaturation. Additionally,the non-ablation settings of the laser have the ability reduce volumeand directionally bend or shrink cartilage, thereby effectively reducingthe volume of a bulging disc. This benefit allows reduction of a bulgingdisc away from any juxtaposed spinal nerves.

Second, the non-ablation settings also allow one to form temperospatialbubbles or waves that create micro and nano pores due to the mechanicalnature of their rapid formation and resolution. These micro and nanopores increase water permeability and thus, aid disc rehydration and theflow of nutrients.

Finally, the direct mechanobiologic effect of the temperospatial bubblesor waves particularly when the laser is in pulse mode, stimulates discchondrocyte proliferation and production of new cartilage.

Injection of Stem Cells/Disc Chondrocytes/PP/RP and Scaffold to TreatDisc Nucleus

After photoactivation, one may inject the composition for regenerationinto several areas of the disc nucleus pulposus and/or into theposterior/posterolateral annulus fibrosus under fluoroscopic guidance.By way of a non-limiting example, the composition for regeneration maycomprise, consist essentially of, or consist of 1-5 cc BMAC, 0.1-20 mm³chondrocyte nucleus material and scaffolding and 0.5 cc-5 cc PRP or PPP.Optionally, one may use a biologic scaffold made from the patient's ownplasma to treat the annulus fibrosus and nucleus pulposus of therecipient disc levels(s) and the donor disc level as well. In someembodiments, the scaffold for disc regeneration may comprise, consistessentially of or consist of one or more, if not all, of PRP or PPPhyaluronic acid, fibrin glue that may for example be formed fromfibrinogen and thrombin, and a flowable wound matrix, such as isavailable from Integra or Matristem. The scaffolding may comprise,consist essentially of or consist of one or more if not all of thefollowing: Type I collagen, Type II collagen, proteoglycans, andglycosaminoglycans. To obtain the scaffolding, one may for example,begin with 5-20 pieces, e.g., 10 pieces of approximately 0.5-2.5 mm³e.g., 1 mm³ in size, morselize them and combine with 0.1-2 cc sterilesaline, e.g., 0.5 cc sterile saline with antibiotics. In someembodiments, 1-4 cc or 2-3 cc of scaffolding is inserted.

As noted above, in addition to generation of platelet rich plasma fromblood for the composition for regeneration, the centrifugation processwill result in the formation of two additional strata. The platelet poorplasma from that strata may be used a stem cell scaffold, and also maybe effective at reducing pain. The platelet poor plasma's ability to actas a scaffold is due to the presence of a network of concentratedproteins. Beneficially, the scaffold is an autologous degradablescaffold that can hold together and support bone-marrow-derivedmesenchymal stem cells and/or adipose-derived perivascular stem cells.

The fibrinogen may be used as a fibrin sealant, and the fibrinogen maybe converted to fibrin by the use of thrombin. The resulting thrombinclot that is formed is used as a sealant in order to fill degenerativeannular tears and to seal a stem cell introduction site or herniateddisc annulotomy site. Thus, the fibrinogen may also be part of thescaffold.

The scaffold may serve one or more of the following functions: (1) toprovide a matrix for stem cells to integrate/engraft during tissuetransplantation; (2) to provide a platform for cell differentiation andcell to cell signaling; (3) to provide an osmotic pump to restoreextracellular matrix hydration; (4) to fill gaps/tears in disc structurein order to restore/normalize disc function; and (5) to stabilize discintegrity.

PP/RP Treatment of Disc Annulus

In order to regenerate a disc, preferably one re-establishes thehydraulic effect of the disc. Using a scaffold, one can essentially sealthe disc and thereby re-establish high intradiscal pressures.Accordingly, to the annulus fibrosus of the recipient disc level, onemay apply PP/RP in order to heal annular tears and to filldefects/tears. Application may for example be to an epidural or a facetjoint region.

When treating the disc annulus fibrosis with PP/RP, one may inject thePP/RP into any area of the annulus fibrosus for example, the posterior,posterolateral or anterior regions. This may be done with or without ascaffold comprising, consisting essentially of, or consisting of CaCl₂,hyaluronic acid, BMP, hydrogel, PLGA or poly (lactic-co-glycolic acid).In one embodiment, the PP/RP may be combined with calcium chloride at aratio of 0.90-0.99 cc (e.g., 0.05 cc) PP/RP to 0.10-0.01 cc (e.g., 0.05cc) of coagulant solution (e.g., 10% CaCl₂+5000 IU thrombin).Optionally, there is also a radiopaque dye.

Treatment of Donor Disc

Optionally, one may apply BMAC and PP/RP to the donor disc level inorder to cause regeneration at the site from which the chondrocytes weretaken. Because the donor disc level already has healthy discs, the BMACand PP/RP do not need to have been combined with chondrocytes prior tointroduction. Applications may be made to the disc nucleus and annulusin order to reduce the chance of iatrogenic degeneration and to maintainhigh intradiscal pressure within the normal disc by sealing the annuluswithin the scaffold.

As persons of ordinary skill in the art recognize, based on theforegoing, either one or both of the recipient disc level and donor disclevel may be treated: (1) with or without hemoconcentration of BMAC; (2)with or without decompression of nucleus pulposus; (3) with our withoutinjection of regenerative cells into nucleus pulposus and annulusfibrosus; (4) with our without scaffold; (5) with or without sealing ofthe annulus; and (6) with our without donor disc cell transplantation.

Additional Adjuncts

The mixtures that are used to treat the degenerated disc or the donordisc may also comprise one or more of the following: (1) genipin toaugment disc repair; (2) simvastatin (Zocor) to prevent discdegeneration; (3) glucosamine/chondroitin sulfate/DSMO to restore thedisc; (4) extracellular matrices including basement membrane matricessuch as matristem to replace ECM and to promote healing; (5) type I ortype II collagen; (6) insulin-like growth factor-1 (IGF-1); (7) bonemorphogenic protein(s); (8) fibrin microthreads for the scaffold topromote stem cell growth; (9) nanotubes to stimulate and to promote stemcell differentiation; (10) hydrogel(s); (11) LIM mineralization protein(LMP)-1 to increase aggrecan synthesis; (12) Link-N Peptide to increaseproteoglycan and matrix synthesis; (13) Sox-9 to increase collagensynthesis and chondrogenesis; and (14) a scaffold that is plasma-basedand contains Atelocollagen, hyaluronic acid and PEG-PLA.

Applications

Various embodiments of the present invention can be used in connectionwith laser neurotomy/rhizotomy. These techniques serve to ablate smallpain fibers and/or to reduce facet arthritis pain.

Various other embodiments of the present invention can be used inconjunction with facet joint thermal ablation. These techniques can beused for one or more of the following purposes: (1) to ablate terminalfacet joint nerve branch networks with a joint capsule; (2) to stimulatecapsule regeneration; and (3) to provide an outside-in ablation ofintercapsular synovitis.

Additionally, various embodiments of the present invention may be usedin conjunction with a transforaminal epidural application. Thisapplication of stem cells may serve one or more of the following: (1) toprovide regenerative cells to posterior posterolateral disc annulus andanterior facet joint capsules; (2) to reduce inflammation in theposterior/posterolateral disc, epidural space and anterior/medial facetjoint capsule; (3) to reduce pain that originates from theposterior/posterolateral disc, epidural space and anterior/medial facetjoint capsules; and (4) to reduce radicular pain and inflammation fromthe existing nerve and traveling nerve of the level of application.

Preferably for a period of at least two years (unless contraindicated)following application of the methods described herein, a subjectundergoes lumbar traction, whole body vibration and/or inversion.Additionally or alternatively, the patient receives oral glucosamine andchondroitin sulfate for a period of at least two years. Still further,and unless contraindicated, in some embodiments, for a period of atleast two years, the subject takes zinc supplements, such as thoseavailable over the counter. An example of a dose of zinc is 50 mg.

Any of the features of the various embodiments described herein can beused in conjunction with features described in connection with any otherembodiments disclosed unless otherwise specified or implicit fromcontext.

EXAMPLES Example 1 Method of Regenerating Discs (Prophetic)

1. Provide a patient with an IV that contains a prophylactic antibiotic.

2. Obtain platelet rich plasma and platelet poor plasma

-   -   a. Draw 60 cc of peripheral blood. One may draw venous blood        into a syringe that contains an anticoagulant.    -   b. Centrifuge the blood in order to separate layers of blood        tissue and to isolate blood plasma.    -   c. Separate blood plasma via syringe aspiration into platelet        rich plasma and platelet poor plasma.    -   d. Optionally lyse platelet rich plasma platelet membrane into a        lysate.

3. Isolate Mensenchymal Cells

-   -   a. Option A: From bone marrow        -   (i) Optionally, sedate patient, e.g., oral or general (in            some embodiments, the patient is not sedated)        -   (ii) Harvest bone marrow            -   1. Option 1: Pelvis                -   a. Make appropriate size skin opening over anterior                    and/or posterior, right and/or left ilium bones;                -   b. Insert a medium to large bone cannula into depth                    of ilium;                -   c. Aspirate 2-7 cc of bone marrow into a syringe                    that is pre-filled with an anticoagulant;                -   d. Rotate the syringe and repeat aspiration(s);                -   e. Change the depth and repeat aspirations(s);                -   f. Harvest 60-500 cc of bone marrow per ilium;                -   g. Upon completion, hold pressure to control                    bleeding from bone.            -   2. Option 2: Vertebra                -   a. Insert a medium to large bone cannula into                    vertebral body, via left and/or right transpedicular                    approach or posterior oblique approach;                -   b. Aspirate 2-7 cc of bone marrow into a syringe                    that is pre-filled with an anticoagulant;                -   c. Rotate the syringe and repeat aspiration(s);                -   d. Change the depth and repeat aspiration(s);                -   e. Harvest 20-400 cc or 50-100 cc of bone marrow per                    side;                -   f. Upon completion, hold pressure to control                    bleeding from bone.        -   (iii) Process bone marrow            -   1. Place bone marrow into centrifuge tube/bucket            -   2. Run single or multiple centrifugation cycles for 9-30                minutes at 2000-4000 RPM or 2500-3500 RPM            -   3. Aspirate mesenchymal tissue layer into treatment                syringe.        -   (iv) Ready bone marrow cells for treatment by placing into a            pre-heparinized syringe after being concentrated.    -   b. Option B: From Adipose Tissue        -   (i) Sedate patient orally, with IV sedation, with general            anesthesia        -   (ii) Harvest adipose tissue            -   1. Option 1: Lipoaspiration                -   a. Under sterile conditions, infiltrate abdominal or                    flank area with conventional mixture of local                    anesthetic and epinephrine;                -   b. Infiltrate subcutaneous region with saline via a                    low pressure infiltration pump;                -   c. Use micro cannulas; harvest 20-500 cc or 50-100                    cc lipoaspirate;                -   d. Isolate adipose derived stem cells via the                    following steps                -    i. Remove fat layer from top of sample,                -    ii. Wash sample to remove local anesthetic and                    epinephrine,                -    iii. Digest with collagenase or emulsify with                    lecithin to remove fat tissue from stromal fraction,                    and                -    iv. Centrifuge to form a stem cell pellet;                -   e. Optionally, segregate a small aliquot of sample                    for cell counting and microscopy for research                    purposes;                -   f. Sample filtration with 40-70 micro nylon cell                    strainer;                -   g. Optionally,                -    i. Combine isolated stem cells with platelet rich                    plasma                -    ii. Sequestor antibodies via IgG magnetic beads                -    iii. Activate stem cells via light            -   2. Option 2: Mechanically                -   a. Ex vivo                -    i. Harvest lipoaspirate as described above or via                    direct excision;                -    ii. Cavitate lipoaspirate to break down lipid from                    SVF (e.g., using ultrasonic laboratory devices such                    as from Hielscher);                -    iii. Centrifuge stem cell pellet;                -    iv. Follow steps (e)-(g) under lipoaspirate                    technique.                -   b. In vivo                -    i. Cavitate ultrasonically and process stem cell                    pellet;                -    ii. Place a vacuum device over an abdominal or                    flank region;                -    iii. Aspirate fat;                -    iv. Process stem cell pellet.

4. Ready stem cells in a syringe that was prewashed with ananticoagulant.

5. Ready platelet rich plasma in a second syringe prewashed withanticoagulant.

6. Ready P platelet rich plasma in a third syringe prewashed withanticoagulant.

7. Harvest disc chondrocytes from a spinal level that is not in need ofregeneration or from the sacrum.

-   -   a. Option A: Donor Spinal Disc        -   (i) Approach intervertebral disc either percutaneously or            open transpedicular or intraspinal.        -   (ii) Harvest nucleus and/or annulus tissue from            intervertebral disc.    -   b. Option B: Sacral Disc        -   (i) Approach S1 sacral disc, percutaneously, or through an            open approach transpedicular or through a trans S1 approach.        -   (ii) Harvest nucleus and/or annulus tissue from sacral disc.

8. Inject autologous mensenchymal stem cells, and optionally plateletrich plasma and optionally platelet poor plasma, into donor disc siteincluding annulus. Optionally, inject platelet poor plasma or hyaluronicacid or fibrin glue or flowable wound matrix into the recipient disc lysate or cytokines or platelet rich plasma into the posterior and/orposterolateral disc annulus.

9. Remove donor disc material (chondrocytes and scaffold) fortransportation.

10. Morselize disc tissue in small aliquot of saline using a scalpel orother mechanical methods, optionally, in conjunction with an extrusionstep.

11. Treat target disc with laser, either unilateral or bilateral. Forexample, one may use an intradiscal Holmium: YAG or Erbium lasertreatment using a non-ablation setting for micro/nano pore formation,disc chondrocyte activation and proliferation.

Optionally, steps 8 and 10-11 may be performed outside of the body.

Example 2 Harvesting Bone Marrow for Stem Cell Processing (Prophetic)

Using a Jamshidi needle, harvest 60-400 cc of bone marrow from the iliaccrest. Place the bone marrow into a syringe with an anticoagulant, e.g.,ACD. Process the bone marrow into bone marrow aspirate. The formation ofbone marrow aspirate concentrate from bone marrow may, for example, beperformed by Celling Biosciences (Spine Smith).

Optionally, one concentrates BMAC with, for example, a hemoconcentrator.When generating BMAC from bone marrow, there is a reduction of volume by80-95%. Thus, for every volume of bone marrow, one gets 5-15% of thatvolume of BMAC. By way of example, 60 cc of bone marrow yieldsapproximately 6 cc of BMAC. The Hemoconcentrator can concentrate thisvolume by approximately 40-60% (e.g., 50%) to 1-4 cc or 2-3 cc, e.g.,approximately 3 cc.

I claim:
 1. A composition for regenerating a disc, said composition comprising: (a) stem cells from a subject; (b) donor disc cells, wherein the donor disc cells are derived from a non-degenerative level of a spine of the subject; and (c) a platelet plasma composition.
 2. The composition of claim 1, wherein the stem cells are mesenchymal stem cells.
 3. The composition of claim 1, wherein the stem cells are derived from at least one type of tissue selected from the group consisting of bone marrow, subcutaneous fat, synovial fluid, peripheral blood, amniotic fluid and amniotic membrane.
 4. The composition of claim 3, wherein the stem cells are derived from at least two different types of tissue selected from the group consisting of bone marrow, subcutaneous fat, synovial fluid, peripheral blood, amniotic fluid and amniotic membrane.
 5. The composition of claim 4, wherein the stem cells are derived from bone marrow and subcutaneous fat.
 6. The composition of claim 1 further comprising red blood cells.
 7. The composition of claim 1, wherein the donor disc cells have been morselized.
 8. The composition of claim 1, wherein the platelet plasma composition comprises platelet rich plasma.
 9. The composition of claim 1, wherein the platelet plasma composition further comprises platelet poor plasma derived from bone marrow.
 10. The composition of claim 1 further comprising platelet-derived growth factors.
 11. The composition of claim 1 further comprising cytokines.
 12. The composition of claim 10 further comprising calcium chloride.
 13. The composition of claim 12 further comprising thrombin.
 14. The composition of claim 11, wherein said composition has been photoactivated.
 15. The composition of claim 1 further comprising a biologic scaffold.
 16. The composition of claim 15, wherein the biologic scaffold comprises a substance selected from the group consisting of plasma, a hyaluronic acid-based material, atelocollagen, PEG-PLA and a hydrogel.
 17. The composition of claim 1 further comprising at least one of genipin, simvastatin, a combination of glucosamine/chondroitin sulfate/DSMO, matristem, collagen, insulin-like growth factor-1, bone-morphogenic protein, fibrin microthreads, nanotubes, LIM mineralization protein (LMP)-1, Link-N-Peptide, Sox-9 and a stromal vascular fraction.
 18. A process for creating a composition for disc regeneration, said process comprising: (a) morselizing donor disc material, wherein the donor disc material comprises healthy cells obtained from a non-degenerative level of a spine; (b) extruding the donor disc material to form an extruded material that comprises chondrocytes and an extracellular matrix; and (c) mixing the chondrocytes, the extra cellular matrix, a bone marrow aspirate, wherein the bone marrow aspirate comprises stem cells and a platelet plasma composition, wherein the platelet plasma composition comprises growth factors to form a composition for disc regeneration.
 19. The process according to claim 18, wherein the bone marrow aspirate further comprises red blood cells.
 20. The process according to claim 18, further comprising mixing calcium chloride in step (c).
 21. The process according to claim 20, further comprising mixing with thrombin in step (c).
 22. The process according to claim 18, further comprising photoactivating the stem cells.
 23. The process according to claim 18, further comprising sequestering platelets from whole blood or bone marrow in order to obtain the platelet rich plasma.
 24. The process according to claim 23, further comprising extracting and concentrating the platelets into a platelet lysate through a cell membrane lysis procedure that uses at least one of thrombin and calcium chloride.
 25. The process according to claim 18, further comprising combining the composition for disc regeneration with a biologic scaffold, wherein the biologic scaffold comprises at least one substance selected from the group consisting of platelet poor plasma, hyaluronic acid, fibrin glue and a flowable wound matrix.
 26. A process for regenerating a disc, said process comprising: (a) harvesting bone marrow from a person; (b) generating bone marrow aspirate and a platelet plasma composition from the bone marrow, wherein the bone marrow aspirate comprises mesenchymal stem cells, plasma, platelets and red blood cells; (c) harvesting disc chondrocytes and an extra cellular matrix from non-degenerative disc tissue of the person; and (d) combining the bone marrow aspirate and the disc chondrocytes to form a composition under conditions that permit the mesenchymal stem cells to begin differentiation.
 27. The process according to claim 26, further comprising photo-activating the composition.
 28. A method for treating a subject in need of regeneration of a disc comprising administering a therapeutically effective amount of a composition for regeneration according to claim 1 to the subject.
 29. A method for treating a subject in need of regeneration of a disc comprising claim 26 and further comprising administering the composition and a scaffold to the subject. 